Unpredictable rainfall, increases in soil salinity, and low temperature at the beginning or end of the growing season often result in decreased plant growth and crop productivity. These three environmental factors share at least one element of stress and that is water deficit or dehydration.
Drought is a significant problem in agriculture today. Over the last 40 years, for example, drought accounted for 74% of the total U.S. crop losses of corn (Agriculture, U.S. Department of, 1990. Agricultural Statistics. US Government Printing Office, Washington, D.C.). To sustain productivity under adverse environmental conditions, it is important to provide crops with a genetic basis for coping with water deficit, for example by breeding water retention and tolerance mechanisms into crops so that they can grow and yield under these adverse conditions.
When the rate of aspiration exceeds that of water uptake or supply, water deficit occurs and wilting symptoms appear. The responses of plants to water deficits include leaf rolling and shedding, stomata closure, leaf temperature increases, and wilting. Metabolism is also profoundly affected. General protein synthesis is inhibited and significant increases in certain amino acid pools, such as proline, become apparent (Barnett et al., Plant Physiol. 41, 1222 (1966)). During these water deficit periods, the photosynthetic rate decreases with the ultimate result of loss in yield (Boyer, J. S., In: Water deficits and plant growth, T. T. Kozlowski (ed.)., Academic Press, New York., pp. 154-190 (1976)). If carried to an extreme, severe water deficits result in death of the plant.
Several mechanisms appear to enable water deficit-tolerant plants to survive and produce. For example, a comparison of drought-resistant and drought-sensitive lines of Zea mays indicates that higher levels of abscisic acid (ABA), which is known to regulate stomata opening and perhaps other signal responses are correlated with resistance (Milborrow, B. V., In: The physiology and biochemistry of drought resistant plants, Paleg and Aspinall (eds.), Academic Press, N.Y., pp.348-388 (1981)). In addition, ABA-insensitive mutants and ABA-deficient mutants of Arabidopsis are prone to wilting (Koorneef et al., Theoret Appl Genet., 61, 385 (1982); Finkelstein et al., Plant Physiol. 94, 1172 (1990)).
Of the mechanisms employed by water deficit-tolerant plants to grow and yield, those with major impact on plant productivity are osmotic adjustment through the increased synthesis of osmoprotective metabolites, control over ion uptake and partitioning within the plant, ability to increase water intake, and acceleration of ontogeny. Examples of osmoprotective metabolites include sugars, such as sugar alcohols, proline, and glycine-betaine (Bohnert et al., The Plant Cell, 1, 1099 (1995); McCue et al., Tibtech, 8, 358 (1990)). Sugar alcohols, or polyols, such as mannitol and sorbitol, are major photosynthetic products of, and are known to accumulate to high levels in, various higher plant species. While mannitol is the most abundant sugar alcohol in at least 70 plant families, it is not produced at detectable levels in any important agricultural field or vegetable crop, other than celery (Apiaceae), coffee (Rubiaceae), and olive (Oleacea). Other sugar alcohols, such as ononitol and pinitol, are known to be produced in some plants under conditions of stress from drought, salt, or low temperature.
To produce a plant with a genetic basis for coping with water deficit, Tarczynski et al. (Proc, Natl. Acad. Sci. USA, 8, 2600 (1992); WO 92/19731, published Nov. 12, 1992; Science, 225, 508 (1993)) introduced the bacterial mannitol-1-phosphate dehydrogenase gene, mtlD, into tobacco cells via Agrobacterium-mediated transformation. Root and leaf tissues from transgenic plants regenerated from these transformed tobacco cells contained up to 100 mM mannitol. Control plants contained no detectable mannitol. To determine whether the transgenic tobacco plants exhibited increased tolerance to water deficit, Tarczynski et al. compared the growth of transgenic plants to that of untransformed control plants in the presence of 250 mM NaCl. After 30 days of exposure to 250 mM NaCl, trangenic plants had decreased weight loss and increased height relative to their untransformed counterparts. The authors concluded that the presence of mannitol in these transformed tobacco plants contributed to water deficit tolerance at the cellular level.
While Tarczynski et al. (WO 92/19731, published Nov. 12, 1992)) disclose that the same methodology might be applied to other higher plants, such as field crops, the introduction of exogenous DNA into monocotyledonous species and subsequent regeneration of transformed plants expressing useful phenotypic properties has proven much more difficult than transformation and regeneration of dicotyledonous plants.
Thus, there is a need for transgenic monocot plants that are resistant or tolerant to a reduction in water availability. Also, a method to produce transgenic monocot plants with increased levels of osmoprotectants is needed.